During infections of humans, several gram-negative bacteria deliver virulence factors to the host cell cytoplasm, employing a specialized export mechanism, called the type III secretion pathway. Yersinia attach to the surface of macrophages and target cytotoxic Yop proteins directly into the eukaryotic cytosol. Other Yop proteins are also secreted from the bacteria by the type III pathway, and target the Yops to sites beyond the plasma membrane of the host cell. The PI has identified a novel targeting mechanism that allows Yersinia to distinguish proteins for either secretion or targeting. An mRNA signal has been identified which is necessary for coupling secretion to targeting during secretion, while the second mechanism involves binding of a Syc chaperone to a specific domain on the Yop protein, resulting in targeting of the Yop protein to the cytosol. The molecular mechanism of recognition and targeting will be studied in this proposal using genetic and biochemical methods. Furthermore, an in vitro secretion and targeting system will be developed. If successful, the information gained from this research may provide new targets for antimicrobial therapy.